Fig. 2: TDP-43 mRNA localization and local translation in MN axons.

a, Quantitative RT–PCR analysis of the relative mRNA levels of TDP-43 and PolB in RNA preparations from soma or axonal compartments of radial MFCs. n = 3 neuronal cultures; each repeat represents a pool of several radial chambers. RQ, relative quantification. NS = 0.9814, **P = 0.0015. b, Representative image of an agarose gel with cDNA amplicons of TDP-43, PolB and β-actin amplified from sciatic nerve axoplasm RNA. n = 5 mice. c, Representative images of smFISH for mRNA of TDP-43 and β-actin in primary MN axons compared to no probe control. Scale bar, 5 µm. d, Representative images of smFISH for mRNAs of TDP-43 and β-actin in EDL muscle NMJs compared to no probe control. White indicates smFISH–NFH three-dimensional co-localization result. Scale bar, 10 µm. e,f, Representative images and quantification of TDP-43 puro-PLA in axons in the presence or absence of anisomycin (40 µM) and in in vitro NMJs (lowest panel). Scale bars, 20 µm and 5 µm. n = 97, 91, 62 axons and NMJs, respectively. NS = 0.0525, ****P = 3 × 10⁻¹⁵ (Aniso), P = 1.31 × 10⁻⁶ (NMJ). g,h, Representative images and orthogonal slices of TDP-43 puro-PLA in EDL NMJs treated with puromycin (g) or with anisomycin + puromycin (h) muscles. i,j, Representative images and orthogonal slices of β-actin puro-PLA labeling in EDL NMJs treated with puromycin (i) or with anisomycin + puromycin (j) muscles. SynP, synaptophysin labeling. Scale bar, 10 µm. For a, data are shown as the mean ± s.e.m., two-way ANOVA with multiple comparisons. For c, representative experiment repeated in three neuronal cultures. For d, representative experiment repeated in three ex vivo muscle preparations. For f, data are shown in violin density plots with markings of first, median and third quartiles, one-way ANOVA with Holm–Sidak correction for multiple comparisons, repeated in three neuronal cultures. Aniso, anisomycin; coloc, co-localization; NS, not significant.

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